1. Studies of Cellular Processes in Living Cells
Mammalian cell culture is an essential tool for fundamental research in eukaryotic biology and it has contributed to advances in virology, somatic cell genetics, endocrinology, carcinogenesis, toxicology, pharmacology, immunology and developmental biology (McKeehan, W. I., In Vitro Cell Dev. Biol., 26, 9-23, (1990)). Classical cell culture technology is carried out in nutrient mixtures with cells usually cultured as a monolayer attached to a hydrophilic surface, commonly sterile treated polystyrene. Considerable progress has been made in developing cell culture systems for specific cell types, with the aim of reconstructing the cell and its environment into a defined unit for the study of responses and properties of cells in a dynamic context. However, experimentation on such culture systems using biological assays is often limited by the need to use invasive or disruptive processes that compromise the structural and functional integrity of the cells.
Certain types of investigations lend themselves particularly to studies with whole cells and inevitably require cell culture techniques as an essential step in the investigation. General areas of study include:
(i) intracellular activity, including the replication and transcription of nucleic acids, protein synthesis and lipid metabolism, PA1 (ii) intracellular flux, i.e. movement of RNA from cell nuclei to the cytoplasm, translocation of human receptor complexes, fluctuations in lipid and protein metabolic pools, transport of ions and other small molecules across membranes, PA1 (iii) environmental influences, including nutrition, infection, virally or chemically induced transformation, drug action and metabolism, response to external stimuli and secretion of specialized products, and PA1 (iv) cell-cell interaction, including embryonic induction, cell population kinetics, cell-cell adhesion and motility.
A vast array of radiolabelled ligands which are available commercially, has played a major role in the development of methods currently used to study intracellular activity, metabolism and cell-ligand interactions in cell culture assay systems. Particular examples relating to the study of cellular processes are:
Thymidine Uptake
Studies involving the measurement of [.sup.3 H]thymidine uptake currently suffer from an absolute requirement for cell disruption and consequently are prone to artifactual effects (Adams, R. L. P., Cell Culture for Biochemists, p 181-192; Saegusa, Y. et al, J. Cell Physiol., 142, 488-495 (1990)). In addition to providing an assessment of cellular proliferation and growth in living cells, thymidine uptake studies are also used to study the extent of DNA repair and/or damage occurring during culture, in the presence or absence of external agents (McKeehan, W. et al, In Vitro Cell Dev. Biol., 26, 9-23, (1990)). Current methods however, require cell disruption and do not readily lend themselves to temporal studies. Thymidine uptake has been used more recently, in tandem with other potential markers, in the field of programmed cell death, or apoptosis, where there is currently considerable pharmacological and clinical interest (Tritton, T. and Hickman, J., Cancer Cells Quarterly Rev., 2, 95-105, (1990)). However, few of the current methods are able to explore and quantify spatial and temporal events occurring during apoptosis (Lock, R. B. and Ross, W. E., Proc. Amer. Assoc. Cancer Res., 30, 621, (1989)). [.sup.3 H]Thymidine uptake studies are also used in cell cycle studies in order to monitor regulation of this essential process (Studzinski, G. P., Cell Tissue Kinetics, 22, 405-424, (1989)). However, there are currently no methods available for the direct measurement of thymidine uptake in living cells.
Receptor Binding/Kinetic Studies
Most of the methods used in this field require binding of a radiolabelled ligand, followed by quantification of receptor number and affinity in competition studies at a fixed time (Goldstein, J. L. and Brown, M. S., Methods in Enzymol., 98, 241-260, (1985); Zoon, K. C. et al, J. Pharmaceutical and Biochemical Analysis, 7, 147-154, (1989)). These methods often utilize membrane filter assays in vitro. The majority of methods require release of cells from a monolayer and often necessitate isolation of cell membranes. These systems are therefore not suitable for real time kinetic studies. Thus in the cytokine field, where specific ligand-receptor binding studies are of fundamental importance, it is not possible to monitor binding, uptake and internalization of specific radiolabelled ligands, as a function of time in living cells (Rakowicz-Szulczynska, E. W., et al, J. Immunol. Methods, 116, 167-173, (1989)). Currently disruptive techniques are required to differentiate between these important processes. This is also the case for studies of receptor cycling, an important process during the receptor-mediated endocytosis of a variety of essential ligands (Anderson, R. G. W., et al, Cell, 10, 351-364, (1977)).
Lipid Metabolism
Studies on the regulation of lipid biosynthesis are usually limited by the disruptive experimental procedures required to determine the incorporation of radioactively labeled lipid substrates. Such experiments are generally performed under optimal conditions in vitro, that may not reflect the situation in vivo, due to an inability to measure variations, both temporally and spatially, in living cells (Vance, D. E. and Vance, J. E., Biochemistry of Lipids and Membranes, pp.116-120, (1989)). The Hep G2 human tumorigenic cell line is currently widely used to investigate lipid and lipoprotein metabolism. Pulse-chase studies are currently difficult to perform when using radiolabelled precursors such as oleic acid, as a function of time. This is because there is a requirement for disruption of the cell in order to differentiate localized areas of uptake. To date, the only metabolic studies that can be carried out with living cells have used fluorescently labeled lipids (Pownall, H. J., Chem. Physics of Lipids, 50, 191-212, (1989)). There are, however, inherent problems with such studies since the fluorophors used tend to be extremely bulky relative to the lipid. The physiological integrity of such labeled lipids is therefore questionable and they are known to be taken up in Hep G2 cells at different rates and incorporated differently within the cell, relative to unlabelled lipid (Pownall, H. J.). Similar problems are also encountered in studies related to protein uptake and metabolism as seen for example when pulse-chase studies using radiolabelled methionine and/or leucine are carried out (Freshney, R. I., Culture of Animal Cells: A Manual of Basic Technique, 2nd Edition, Alan R. Liss Inc. (1987), pages 227-236). Unless fluorescent probes are used, disruption of the cell is always necessary (Smith, L. C., et al, Methods in Enzymol., 129, 858-873, (1986)).
Cell Calcium
Calcium concentrations in the cytoplasm are carefully regulated by several mechanisms and the affinity for calcium and its rate of transport across the membrane, vary considerably from cell to cell. In early cellular studies, calcium sensitive fluorescent dyes were injected invasively, thus restricting their usefulness in kinetic measurements. The more recent use of dyes such as Fura-2 has alleviated this problem, although quantitative, time-dependent measurements of calcium are still difficult to carry out in culture (Cavaggioni, A., Bioscience Results, 9(4), 421, (1989)). Alternatively, [.sup.45 Ca] (Kuwata, J. H. and Langer, G. A., Molecular Cell Cardiology, 21, 1195-1208, (1989)) and Langer, G. A. et al., Circulation Res., 24, 589-597, (1969)) and .sup.42 K (Frank, J. S. et al, Circulation Res., 41, 702-714, (1977)) exchange has been measured in cardiac cells. Using sterile scintillant coated discs in a flow cell chamber, neonatal rat hearts were cultured and the time dependent uptake of [.sup.45 Ca] and [42 K] was monitored by pulse-chase methods. Although time dependent measurements in living cells are recorded in this study, there are several disadvantages with this system: (i) the system is only applicable to one sample at a time, (ii) large quantities of [.sup.45 Ca] are used for uptake studies, (iii) there is a high background/non-specific signal and (iv) discs are removed from the sterile culture medium and exposed to air before insertion into the flow cell. There is therefore no opportunity to perform additional measurements or to continue to culture the sample.
Consequently, there is still a requirement, not only in this case for calcium uptake measurement, but also in the processes described previously, for non-invasive, non-disruptive, real-time whole cell measurements in a format which is amenable to high sample throughput. The invention described here is intended to overcome the problems and limitations of the prior art methods and will greatly facilitate the above objectives.
2. Developments in Scintillation Counting Technology
Detection of receptor binding or cellular metabolic events utilizing radiolabelled substrates is accomplished by scintillation counting, usually following extraction or separative procedures, which are generally laborious, time consuming and are not amenable to automation.
A means for overcoming such problems is described in U.S. Pat. No. 4,568,649 (Bertoglio-Matte). This covers an homogeneous assay procedure which produces quantifiable light energy at a level which is related to the amount of radioactively labeled reactant in the assay medium. The light energy is produced by a scintillant which is either incorporated, or forms part of, a support structure (beads or other solid surface which can be used in the assay process). The support structures are coated with a receptor or other capture molecule, and are therefore capable of specifically binding the radiolabelled ligand or reactant of interest. In a direct assay, a sample containing the reactant is mixed in aqueous solution containing scintillant support bodies to which a binding compound may be attached. The reactant is caused to bind with its corresponding binding compound, thereby placing the radiolabelled species in close proximity to the scintillant-containing support. The scintillant is activated causing emission of light, which can be detected conventionally using a scintillation counter. The amount of light produced is directly proportional to the amount of reactant bound to the surface of the support structures.
Ideally the isotope of the radiolabel should have a relatively low energy beta-emission, for example tritium, or iodine-125 auger electrons. Only that portion of the sample which binds to the binding molecule, and is therefore in close proximity to the scintillant will result in scintillation events that can be counted. Unbound reactant will be at too great a distance from the scintillant surface to produce scintillations, the beta-decay energy being dissipated in the liquid aqueous medium.
A considerable advantage of the scintillation proximity assay process is that it does not require separation of bound molecular species from free. Such a process will also minimize the need to handle potentially hazardous/radioactive substances, as well as being more convenient and amenable to automation.
In U.S. Pat. No. 4,568,649, capture molecules are attached to, and fluorescer is integrated into beads, for example polyacrylamide beads. The Scintillation Proximity Assay technique may also be performed with other types of support structure. European Patent Application No. 0378059 describes a support structure for scintillation proximity assays comprising a fibre mat which incorporates a fluorescer. In one format the fibre mat consists of solid scintillant forming a matrix. The scintillant can be a cerium loaded glass or may be based in rare earths such as yttrium silicate (with or without activators such as Tb.sup.3+, Eu.sup.3+, Li.sup.+). The scintillant fibre may also be composed of a scintillant polymer such as polyvinyltoluene. As an alternative, an organic scintillant such as 2,5-diphenyloxazole (PPO) or anthracene may be coated onto a fibre mat which is made from non-scintillant material. The fibre mesh format presents a large surface area upon which binding reactions can occur.
PCT Application No. WO 90/03844 discloses a microtitre well plate intended for binding assays. There is no claimed application for living cell-based assays. The sample plate may be produced from a transparent scintillant-containing plastic by means of a vacuum thermoforming or injection moulding process. In principle the walls of the plate may be coated with binding compound for the purpose of carrying out in vitro binding assays using radiolabelled reactants. However no practical examples are given in the application. It is possible, for example, that one disadvantageous effect of using a plate made from clear plastic will be that light generated in one well of the plate may be detected in adjacent wells, a phenomenon known as "cross talk", thereby causing high assay backgrounds and spurious assay results. The plates are not described as being treated in any way to support cell culture or growth.
Burton, J. A. and Hoop, B. describe a method and apparatus for ligand detection (PCT Application No. WO 88/04429). Central to the process is a reaction chamber and sensor surface connected optically to a detector. In a typical format of the method, a sample containing the ligand to be measured is introduced into the chamber containing receptor molecules immobilized on the sensor surface, and radioactively labeled ligand molecules. As a result, a portion of the labeled ligand molecules is displaced from the surface causing a decrease in fluorescent events at the sensor surface. However, the apparatus described in this application is designed for continuous throughput competitive binding assays. There is no reference to, or applications in, the study of living cells.
In summary, none of the prior art methods published for SPA are amenable to the study of biochemical processes in living cells. In part, this is due to the fact that the current fibre and bead based technologies are not suitable for monolayer cell culture. The only scintillant plate format so far described is intended for in vitro radiolabelled binding assays, and furthermore has inherent potential disadvantages outlined above for the specific applications which are the subject of this invention.